The Crystallisation Of Hen Egg White Lysosyme Biology Essay

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The lysosyme crystal is a relatively easy protein to crystallise and the range of conditions at which it can crystallise is evidence to this statement. In respect to this, knowledge of the solution conditions aids in creating suitable condition niches for future crystallisation of this protein and other related proteins. The factors varied in the two weeks grown crystals include pH: 4.5, 5.5, 6.5, 7.5, 8.5, varied using the buffer solutions, and the NaCl precipitant concentration, M: 0.85 and 1.90. Different buffers were also used: 0.1M NaAcetate, 0.1M MES, 0.1M Hepes and 0.1M Tris-HCl. Crystals formed in most of the conditions except in columns 3,4,7,11,12. Columns 1 9, 10 produced equally the greatest number of well-ordered diffractable crystals. Results revealed the widely observed randomness in crystal formation in which even in constant conditions crystals do not always form.

INTRODUCTION

Macromolecular crystallisation of proteins have greatly advanced modern science and have helped greatly understand the function of proteins and their collective functions in the human body which has been essential to the design of drugs with greater efficacy. However the amount work and difficulties that has been undergone to grow and image the crystals which are then used to generate 3D images are not as recognised as well as the results. Due to the diversity of proteins, crystallisation of protein's still proves to be a challenge even with the relatively vast increase in science and technology. Precipitant concentration, temperature, pH, interfacial tension and super-saturation of protein solution are factors which have effects on the nucleation rates of the components of the crystal which is a required preliminary stage of protein growth and crystallisation.

Prior to protein growth and crystallisation the protein has to first be produced, extracted and purified to a 96% purity level. 96% is a general value as some proteins cannot be purified to this level. Advances in genetic engineering have provided highly efficient methods to manufacture very high quality proteins and effective methods of testing the purity of the protein such as SDS electrophoresis. When the satisfactory level of purity is achieved after the protein is ready to undergo crystallisation. Hen egg lysosyme is an example of a protein which has been crystallised on many occasions. Crystallisation is basically the removal of water from the protein solution but in such a way that it generates a rigid protein structure due to the amount and orientation of well ordered and disordered water molecules which are bound, surround and hold the proteins crystal lattices together via comparatively weak hydrogen bonds and van der Waals forces. From knowledge of the crystal lattice structure and general chemistry it can be obviously deduced that protein crystal when in comparison to inorganic salt crystal are of greater fragility. For this same reason the sizes of protein crystals are rarely ever greater than 1mm in their shortest dimension. Rhodes et al states that 'Protein crystallography requires a crystal of at least 0.2mm', [4].

The growth of crystal is via a method called the sitting drop technique. The diagram below illustrates the setup. The crystals form as a result of the phenomena of diffusion and can be fully explained using Chatelier's principle which states that :'If a chemical system at equilibrium experiences a change in concentration, temperature, volume, or partial pressure, then the equilibrium shifts to counteract the imposed change and a new equilibrium is established'.

When the two solutions are placed in a controlled environment they will interact reach a point of equilibrium where net movement of water is zero. The reservoir solution is made to be supersaturated so prior to the equilibrium point the net movement of water is into the reservoir solution. So basically water evaporates from the protein solution at a slow rate which encourages growth of large crystals and condenses into the reservoir solution. The reservoir solution maybe changed few times with the aim of removing more water molecules. The lifetime crystal growth varies depending of the physical and chemical properties of the protein. A week is sufficient time to grow a lysosyme protein crystal.

Left -Diagram of the Sitting Drop method [1]. The net movement of water is from the red protein solution to reservoir solution blue via evaporation and diffusion. The gradual removal of water causes a favourable orientation of the water surrounded protein molecules which result in crystal lattice formation.

Water content of crystal is an essential part of the protein crystallisation procedure because to maintain the protein crystal integrity it has to be surrounded by water molecules. As stated in Rhodes t al early crystallographers failed to achieve diffraction patterns from dried protein crystals. The reason for this was later discovered by J.D Bernal and Dorothy Crowfoot when they 'measured diffraction from pepsin crystal in the mother liquor'. As a result of the discovery, X-ray diffraction is carried out in humid atmosphere or whilst the protein is still in the mother liquor. 'The electron-density maps from X-ray data collection reveals many ordered molecules on the surface of crystalline proteins. Disordered water is presumed to occupy regions of low density between ordered particles'. Although the number of water molecules varies from any one protein crystal to the next, the number of 'detectable ordered water molecules averages about one per amino-acid residue in the protein. The ordered water molecules which on an electron density map appear as 'small regions of disconnected density are of great importance to a crystallographer because assignment of the water molecules to isolated areas of the electron density map improves the overall accuracy of the model'.

Crystallisation rarely affects the structure or function of the protein but due to the slight chance crystallisation may affect the protein structure effort must be applied to show that the crystallised protein is not affected. This is done with the knowledge that the protein crystals maintain original functions of the protein. The protein substrate can be added to some of the protein and if product or expected biochemical reaction takes place then it can be assumed that the structure of the protein is maintained as structure is tightly related to structure especially with biological molecules. When this part of the crystallisation process is complete we can be assured that the model of the protein determined is in the functional form.

When crystallising proteins, the pure form, derivative form in which another molecule is bound are required in order to carry out isomorphous replacement. The derivative form is used to obtain phases for the generation of the protein model. Molecule bound proteins are normal as most proteins complex with many other ligands in the body and this aspect of the protein structure needs to be studied. Examples of ligands include inhibitors, substrate and cofactors and allosteric bound molecules. A particular form of derivative crystal is the heavy-metal or ion bound crystal structure which increases the intensity of the diffraction spots. It is also crucial the symmetry structure and unit cell-dimensions remain the same as the diffraction spots will be all put onto one image plate for interpretation.

Unlike inorganic crystal growth, the growth of organic crystal such as that of proteins requires more specific and delicate conditions. The above diagram is one example of the vapor diffusion method in which the protein/precipitant solution is allowed to equilibrate in a closed container with a larger aqueous reservoir whose precipitant concentration is optimal for producing crystals. As the crystal forms the concentration of the protein solution and precipitant solution become equal. As crystallisation is undergone various products can be formed. Examples of these include poor crystalline matter incapable of diffraction called 'sea urchin'. Another type of products formed is intergrown crystals which results from new crystal grow on a preformed crystal. One of the reasons for this is due to the random component of protein crystallisation. Incorrect addition of protein and precipitant solutions results in a product seen as a dark patch when viewed under the microscope. The reason for the dark patch is a result of the crystallising solution lacking one component required to form crystals.

Crystal formation is initiated by the process of nucleation of which there can be two types homogenous and heterogeneous. In homogenous nucleation crystal growth is defined as crystal growth uninfluenced by foreign particles and proteins aggregate from random nucleation events. On the other hand, heterogeneous crystallisation, which is a comparatively faster method than the homogenous form uses various techniques to accelerate the slow earlier stages of crystallisation fundamentally by providing a nucleation site. Examples of methods used for heterogeneous crystallisation include addition of fibrous particles, cuts in the crystallisation container to expose rough edges on which nucleation can occur. The latter method of nucleation is of greater advantage when you know the crystal growth parameters. Micro-seeding is a special heterogeneous technique which uses a piece of crystal from a previously formed to act as a nucleation site.

When attempting to crystallised protein which has not previously crystallised a range of parameters such as temperature, pH, protein and precipitant concentration are explored. It is usually best to 'determine the effect of the pH on precipitation with a given precipitant, repeat this varying the temperatures, and then varying the precipitating agents'. Knowledge of biochemically similar proteins will aid crystallisation as conditions to crystallise these are unlikely to differ greatly. If the protein required to be crystallised is unrelated to already crystallised proteins many trials are likely before a narrowing of the parameter is determined. Robots which prepare solutions and pipette them into containers are very useful and significantly reduce time and effort taken to setup the experiments. The response-surface procedure is a sophisticated scheme for finding optimal conditions for crystal growth. Response surface employs mathematical scoring of results achieved and the 'relationship between parameters and scores are analysed and fitted on a mathematical function. Polynomials which describe a complicated multidimensional surface is an example of this and will highlight peaks where the score is highest and presumed to be the best conditions for protein crystallisation. The graph is manipulated and used to determine the likely conditions which have not undergone trials.

Once a protein has been crystallised it needs to be judged. Characteristics required by the crystallographer include optical clarity, smooth faces and sharp edges. Density used in determination of several useful properties of proteins such as molecular weight and the number of proteins asymmetric unit cell used for electron density map interpretation when solving the structure of the protein should be desirably high. After quality of crystal is determined to be of diffractable quality you can then X-ray image for diffraction patterns to be used for obtaining phase and modulus information.

(My ideas for the introduction came mainly from reference [4] and [6]).

MATERIALS AND METHODS

Materials used;

Oxford design X-ray source-dia-200µm, detector and light microscope

Robotic Mosquito volume dispenser

0.85M,1.90M NaCl

Buffers: 0.1M NaAcetate-pH: 4.5, 5.5. MES-pH: 6.5. Hepes-pH: 7.5. Tris-HCl-pH: 8.5.

Purified Egg white lysosyme solution

Innovaplate SD-2 plate

Gilson p20 pipette

Method:

Pipette 200nl of reservoir solution into larger well with 0.85M NaCl in most columns apart from columns 3-4 which received 1.9M of NaCl.

Place well structure containing NaCl solutions into appropriate place on mosquito machine and allow machine to pipette 200nl of 50mg/ml of lysosyme protein solution into the two smaller wells of innovaplate SD-plate.

Incubate using the sitting drop method for two weeks and analyse crystal using high power light microscope

Record a well order crystal using Oxford design X-ray source detector and in built computer machine.

Machine measures three different angles and three different lengths of the crystal and gives values to 2 decimal places.

Crystal is image at three angles of which x-ray source rotated 0.5o each time.

Diffraction image generated

RESULTS

Table of results

 

1

2

3

4

5

6

7

8

9

10

11

12

A

S

S

S

S

N

N

N

N

N

N

N

N

C

S

S

S

N

N

N

N

N

C

N

N

B

S

S

S

S

N

N

N

N

N

N

N

N

C

S

S

S

N

N

N

N

N

N

N

N

C

S

S

S

S

N

I

N

N

N

N

N

N

C

C

S

S

D

N

N

N

N

C

N

N

D

C

S

S

S

C

N

N

N

N

N

N

N

S

I

S

S

N

C

N

C

N

N

N

N

E

S

S

S

S

N

N

N

N

N

N

N

N

B

B

B

B

B

B

B

B

B

B

B

B

F

S

S

S

S

N

N

N

N

N

C

N

N

S

S

S

S

N

N

N

N

C

I

N

N

G

S

S

S

S

N

N

N

N

C

N

N

N

S

S

S

S

N

N

N

N

C

N

N

N

H

S

S

S

S

N

N

N

N

N

N

N

N

C

C

S

S

I

N

D

N

C

N

N

N

Key

 

Top well

 

Bottom well

S

"Sea Urchin"

C

Crystal

I

Intergrown Crystal

N

No Growth

H

Half Drop (possible mosquito error, no protein)

B

Bad Drop

D

Dust/Fibre (used in seeding)

Crystal unit cell dimension values: lengths a=79.13 Å, b=79.27 Å, 38.94 Å, angles alpha=89.67o, beta=89.83o, gamma=89.62o.

DISCUSSION

From the results it can be seen that 17 well ordered single crystal denoted C in the table were achieved which is a good for a first attempt and because we could generate a relatively good diffraction image from many of them. On the other hand the overall results were poor because we knew the conditions at which hen egg lysosyme crystallises so we should have achieve a greater number of well ordered crystals. The reason we did not achieve this is because even with conditions repeat many times throughout the practical the random nature of the kinetics of protein crystallisation resulted in some wells not forming crystals. A way of getting around this problem is to use seeding techniques which significantly encourages the nucleation and the type which results in crystal formation. As explained in the introduction large single well ordered crystals are desirable as these crystals will give good diffraction patterns of which useful information can be extracted. From the well formed crystal we generated diffraction patterns from which can be combined (if we had carried out derivative crystallisation of our protein) with the derivative pattern and use to solve the phase problem.

From the results table it can be seen that formation of many S denoted, poor crystalline matter incapable of good diffraction pattern occurred. Explanation of this occurrence is again due to the random kinetics of protein crystallisation where many successful nucleation events took place at a fast rate and resulted in formation of many small useless crystals. Temperature cannot be used to explain this as the whole experiment were kept in same temperature conditions The S stands for sea urchin which is the name given to the crystalline matter. The crystal cannot produce diffraction patterns because they are too small and as previously stated a minimum of 0.2mm is the shortest dimension required to work with and these sea urchins are very far from this length. Columns 1-4 had sea urchins but 3-4 had only sea urchins. Although sea urchins cannot be used for diffraction it does provide us knowledge that that conditions which will favour formation of well ordered crystals are close. The difference between columns 1-2 and 3-4 is an increase in the [NaCl] from 0.85M to 1.90M and from more than doubling the precipitant salt concentration the eventualities of random nucleation occurrence increased significantly and produced undesired results.

The whole of row E bottom for every column produced no crystalline matter denoted by B as bad drop and this is likely to have resulted from bubble formation in one of the main volumes of protein solution. This bubble was picked up by the mosquito robot pipette and as a result a smaller volume of the protein solution was dispensed into the wells rather than the wanted protein solution. This confirms that concentration is a factor which affects crystallisation as stated in my introduction, although we cannot be sure that anything was added into the wells at all or the amount which was added. Hence we cannot draw conclusions from the results in this well. Bubbles can be avoided by checking our volumes of solution and by use of standard pipette techniques.

Intergrown crystals were formed in some of the well which is expected. In well 5C bottom a well formed crystal had another badly formed crystal next to it. The badly formed crystal is likely to have risen from a random nucleation event but used the preformed crystal as a nucleation site. The intergrown part of the crystal has a layered and rough appearance likely to have originated as a result of continuous stacking of crystals again results from intergrown crystals on top of the already intergrown crystal but with a different orientation and symmetry. The intergrown crystal described her can be found in the results table at 5C denoted as D because a dust particle was also found there, 5H,6C and 9F

Column 9-10 produced same number of crystal as columns 1-2 but one, 9F was intergrown. I can then conclude that the two conditions were relatively favourable for protein crystal growth even the pH and buffer used was different. Columns 1-2 has Na Acetate buffer whilst columns 9-10 had the Hepes buffer and the respective pH's were 4.5 and 7.5. The mostly likely reason for is could due to the fact that hen egg lysosyme has a wide crystal forming pH range. Another notable occurrence can be seen in the table when you exclude columns 3-4 and 7-12 of which the too many variables where changed to make assured conclusive comparisons. In columns 3-4 and previously stated the concentration was changed and in 7-12 the concentration and pH varied. From columns 1-6 taking into account the exclusion, a gradual reduction in the number of crystals forming reduced as the pH increased from 4.5 to 5.5 which has lead me to conclude that crystal formation decreases with increasing pH for the hen egg lysosyme.

At the same time based on the results I am forced to make a pseudo-contradictory conclusion that at neutral pH of 7.5 crystal formation is also favourable. An explanation which removes the contradiction is that the hen egg lysosyme must have regions in which the protein that are acidic and non polar in nature respectively so encourage can undergo nucleation using different parts of itself.

The rest of the wells had no diffraction and this result is denoted by N in the results table. The reason for this is due to unfavourable crystal growing parameters. Notice how sea urchins were no longer formed at all from column 5 onwards. Single crystals did form in columns 5-6 and 9-10. The other reason for no crystalline matter could be a result of inaccuracy in the direct application of the protein solution in the smaller wells resulting in poor mixing and hence no crystal formation.

We acquired an x-ray diffraction image of crystal 10A bottom. From data analysed from it from the x-ray machine at the following image diffraction angles and axis were: (alpha=89.67, beta=89.83 and gamma=89.62), (a=79.13Å, 79.27 Å and 38.94 Å), respectively. The angles of the crystal having neared 90oand having two very similar axis lengths of 79.13 and 79.27 then one very different axis length of 38.94 it was concluded that the shape of the crystal was tetragonal with a point group symmetry system of 42.The information we had was not sufficient to define the space group but when compared to the data on hen egg lysosyme from the protein data bank website which stated that the unit cell dimensions of the crystal were 79.34 Å, 79.34 Å, and 37.87 Å and angles were all 90o. This solidified the conclusion of the unit cell point group symmetry of P42 and tetragonal shape. The knowledge to estimate what shape and point group was taken from p46 of Rhodes et al which has definition on how to define the six possible shapes for biological molecules which are entiamorphic.

The diffraction pattern we generated alone cannot be used to define the specific space group of the lysosyme crystal because we do cannot define all the different symmetry from just the normal crystal diffraction and this also cannot be used to determine phase information to build the protein crystal model. To determine the space group and solve the lysosyme structure a technique known as isomorphous replacement will need to employed and this requires more experiments and data to be recorded such as the modulus, frequency and phase difference of the diffraction spots on the x-ray image.

To determine the structure heavy atom crystals, (also known as derivative crystals) need to be produced and the data collected for them similar to that of the normal crystal. Heavy atom position diffractions give x-ray diffraction spots of greater intensity in comparison to that of the normal atoms of the protein molecules. The heavy atom derivative crystal will need to anomalously scatter the x-ray beams. Anomalous scatter is scatter with varying wavelength and intensities. The exact positions of the heavy atom derivatives are required and this is accomplished satisfactorily with area detectors. 'The anomalous scatter is used break the phase ambiguity'. The structure amplitude factor and associated phase are described in vector quantities. Once heavy atom positions have been determined the structure factor amplitude and phase angles can be calculated. The following equation relates structure factors of the derivative to that of the native and heavy atom. Fph=Fp+Fh, where Fph, Fp and Fh are the structure factors of the derivative, heavy atom and native protein [6].

The phase is then actually calculated using the following equation Fph2=Fp2+Fh2+2FpFhcos (αp-αh) where αp and αh are the associated phases of the native and derivative structure factors. The vectors are plotted on a vector/argand diagram with imaginary and real axis. The vectors are then actually plotted in complex plane of the diagram. Two results appear when this is carried out and so a second derivative data set is required to determine which of the two the real phase angle is. On the argand diagram the real phase angle is where the three circles intersect on the vector drawn from the factor amplitudes and phase angles. Once the phase problem is solved the values are used along with a Fourier synthesis to compute produce an electron density map of the model which is further interpreted by software program to produce the diagrammatical a three dimensional atomic model and the space group determined. Note that the real phase angle has to be calculated for every edge, hkl of the crystal unit cell.

(My ideas for the isomorphous replacement technique were taken from reference [4] and [6]).

Diagram taken from [2].A harker diagram illustrates how the real phase angle is determined diagrammatically. The point at which the three circles simultaneously intersect is where you will find the real phase angle.

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